Home › Cell Biology › Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
Cell Biology JoVE (Open Access) Citable · DOI

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

DOI: 10.3791/3943-v
What you'll learn
  • ✓ Prepare pooled PCR libraries for targeted sequencing
  • ✓ Align sequencing reads and perform quality analysis
  • ✓ Detect rare genomic variants using SPLINTER software
  • ✓ Interpret SPLINTER output for variant identification
Protocol

Biopharma Insights Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

Difficulty
advanced
Total time
~2 days for library prep and sequencing; ~1 day for analysis

Steps

1
Perform pooled PCR library preparation and sequencing

Prepare pooled libraries from DNA samples of multiple individuals and sequence the targeted cancer-related gene panel using next-generation sequencing.

▶ 01:28
2
Align sequencing reads and analyze coverage

Align the sequencing reads to the reference genome and perform initial quality control and coverage analysis to ensure data suitability.

▶ 04:46
3
Detect rare variants using SPLINTER

Run the SPLINTER software package on the aligned pooled sequencing data to identify rare genomic variants with high sensitivity and specificity.

▶ 07:35
4
Interpret SPLINTER results for variant detection

Analyze the output from SPLINTER to distinguish genuine rare variants from sequencing errors and validate the detected candidates.

▶ 11:11
5
Conclude and evaluate scalability

Summarize the SPLINTER analysis results and discuss the scalability of this method to other phenotypes or larger cohorts.

▶ 13:24
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